1. Field of the Invention
The present invention relates to polarization-based affinity assays.
2. Description of the Related Art
In affinity assays, a known quantity of a labeled probe competes with or binds to an unknown quantity of unlabeled analyte at binding sites on a target molecule for which the analyte has an affinity. The labeled probe that is bound to the target molecule presents a different measurable phenomenon than the labeled probe that is unbound. Calibration curves relate the presence or quantity of the analyte to the relative amount of bound to unbound labeled probe. The calibration curves are generated by measuring the relative amounts of bound and unbound labeled probe in the presence of known quantities of analyte. In competitive binding assays the probe is the same as the analyte or is another entity that competes for the same binding sites on the target molecule. In sandwich binding assays the probe binds to the analyte that is bound to the target molecule. In immunoassays, the analyte is an antigen and the target molecule is an antibody.
In fluorescent affinity assays, the probe is labeled with a fluorophore. A fluorophore is a functional group in a molecule which absorbs electromagnetic waves at a specific wavelength and subsequently emits electromagnetic waves at a different specific wavelength. The electromagnetic waves involved in some fluorophores have wavelengths that are in or near the visible band, and are called light hereinafter.
Polarized electromagnetic waves have a particular directional component for the varying electric field. When linearly polarized light is used to stimulate fluorescence, the light emitted is also linearly polarized. This phenomenon is generally termed fluorescence polarization.
Fluorescence polarization immunoassay (FPIA) is a competitive binding assay that takes advantage of changes in degree of polarization of emitted fluorescent light when a fluorophore-labeled antigen is bound to an antibody. In traditional FPIA, an observable polarization change relies on labeling relatively small antigens with small fluorophores that display relatively long lifetime emissions to obtain depolarized fluorescence from the unbound probe as it rotates in solution at a rapid rate (with a corresponding short rotational correlation time). A small antigen is usually less than 10 kiloDaltons (kDa, where 1 kDa=103 Daltons, and 1 Dalton is a basic unit of atomic weight approximately equal to one twelfth the atomic weight of Carbon 12). An example of a long lifetime fluorophore is Fluorescein with a lifetime of about 4 nanosecond (ns, 1 ns=10−9 seconds). The FPIAs detect the increase in emission polarization when a small probe-fluorophore conjugate binds to a large target molecule that rotates more slowly in solution. The higher the concentration of unlabeled antigen present in a test sample (e.g., from a patient) mixed with the labeled probes, the less bound labeled antigen is present and, consequently, the lower the polarization of the fluorescent light emission.
A disadvantage of the traditional FPIA is the requirement that the labeled probe be much smaller than the target molecules. This restriction eliminates many probes of interest. Since the probes are the same as the analyte in most competitive binding assays, this restriction also eliminates many analytes of interest. For example, molecules of size like streptavidin for which many fluorophore conjugates are commercially available as labels binds to many target biotinylated molecules of interest. Such molecules can not be regarded as conventional polarization probes because of their large size that precludes displaying useful changes in polarization values when bound in solution to biotinylated bovine serum albumin (BSA-bt) as a target molecule. The molecular weight of streptavidin, of about 66 kDa, results in a large rotational correlation time that is too long, when unbound, to be detectably depolarized.